The present invention relates generally to molecular biology and the regulation of cell death and, more specifically, to proteins that modulate the function of the Bax protein, which is involved in inducing apoptosis.
In essentially all self-renewing tissues, a balance is struck between cell production by mitogenesis and cell loss due to programmed cell death, thereby maintaining total cell numbers within a physiologically appropriate range. In pathological conditions, however, the balance in cell production and cell loss can be disrupted. In cancer, for example, an increased amount of cell production due to a shortened cell cycle time or a decreased amount of cell death due to dysregulation of a programmed cell death pathway results in the growth of a tumor.
With regard to programmed cell death, a variety of stimuli, which generally occur external to the cell, initiate a pathway that ultimately results in apoptosis of the cell. As is common for most signal transduction pathways, the various different stimuli that induce apoptosis likely initiate the process of programmed cell death through specific pathways. However, most if not all of these initial pathways converge at a common point that generally involves a member of the Bcl-2 family of proteins.
The Bcl-2 family of proteins regulate a distal step in the evolutionarily conserved pathway for programmed cell death and apoptosis, with some members of this family functioning as suppressors of cell death (anti-apoptotic proteins) and other members functioning as promoters of cell death (pro-apoptotic proteins). Overexpression of the anti-apoptotic protein, Bcl-2, for example, blocks neuronal cell death that otherwise is induced in vitro by various stimuli, including neurotrophic factor withdrawal, various oxidants, glucose deprivation, certain neurotrophic viruses, and amyloid xcex2-peptide. In addition, Bcl-2 is overexpressed in some tumor cells and, in part, may contribute to tumor growth by altering the balance between cell division and cell death.
In comparison, overexpression of the pro-apoptotic protein, Bax, for example, promotes cell death when triggered by a variety of inducers of apoptosis, including growth factor withdrawal, ionizing radiation, and anti-Fas antibody. In addition, elevations in Bax expression occur in association with cell death induced by a variety of stimuli, including neuronal cell death that occurs due to ischemia, epilepsy, spinal cord injury, and certain neurodegenerative diseases such as Parkinson""s disease and Alzheimer""s disease.
Although aberrant expression of members of the Bcl-2 family of proteins is associated with various pathologic conditions, the mechanisms by which these proteins mediate their action is not known. Often, the action of a protein can be inferred from its structural relationship to other proteins, whose functions are known. However, while the Bcl-2 family proteins share certain structural homologies with each other, they do not share substantial amino acid sequence homology with other proteins, further hindering attempts to understand how the Bcl-2 family proteins such as Bcl-2 and Bax regulate cell death. Thus, a need exists to identify proteins involved in the programmed cell death pathway. The present invention satisfies this need and provides related advantages as well.
The present invention provides substantially purified nucleic acid molecules encoding Bax inhibitor proteins, including Bax inhibitor protein-1 (BI-1) and Bax inhibitor protein-2 (BI-2), nucleic acid molecules complementary thereto, portions of such nucleic acid molecules, vectors containing the nucleic acid molecules, and host cells containing the vectors. For example, the invention provides a nucleotide sequence encoding BI-1 (SEQ ID NO: 1) and a nucleotide sequence encoding BI-2 (SEQ ID NO: 4), as well as nucleotide sequences complementary thereto (SEQ ID NO: 2 and SEQ ID NO: 5, respectively).
The invention also provides methods of using nucleic acid molecules encoding Bax inhibitor proteins. Such nucleic acid molecules can be used, for example, as probes to identify the presence of a nucleic acid molecule encoding a Bax inhibitor protein in a sample. A nucleic acid molecule of the invention also can be used to increase the level of expression of a Bax inhibitor protein in a cell by introducing the nucleic acid molecule into the cell under conditions that allow for expression of the encoded Bax inhibitor protein. A nucleic acid molecule of the invention also can be used to decrease the level of expression of a Bax inhibitor protein in a cell by introducing the nucleic acid molecule into the cell under conditions that allow for expression of the molecule in an antisense orientation, such that the antisense molecule can bind to a nucleic acid encoding the Bax inhibitor protein in the cell. By increasing or decreasing the expression of a Bax inhibitor protein in a cell, the likelihood that the cell will undergo apoptosis can be increased or decreased
The invention also provides substantially purified Bax inhibitor proteins. For example, the invention provides a BI-1 polypeptide having the amino acid sequence of SEQ ID NO: 3 (FIG. 1) and a BI-2 polypeptide having the amino acid sequence of SEQ ID NO: 6 (FIG. 2). The invention also provides portions of such polypeptides, which can be useful, for example, for raising antibodies specific for a Bax inhibitor protein. Accordingly, the invention provides antibodies specific for a Bax inhibitor protein such as BI-1 or BI-2.
The invention also provides methods of using a BI-1 or BI-2 polypeptide, or a peptide portion thereof, to identify the presence of other Bax inhibitor proteins or of a member of the Bcl-2 family of proteins in a sample. Such a method is based on the present disclosure that a Bax inhibitor protein can form homodimers and, in addition, can specifically associate with a member of the Bcl-2 family of proteins such as Bcl-2 or Bax to form a heterodimer.
The invention further provides methods of identifying agents that can modulate the formation of Bax inhibitor protein homodimers or that modulate the binding of a Bax inhibitor protein to a member of the Bcl-2 family of proteins. Such a method is useful, for example, for screening large libraries of molecules to identify those agents that can increase or decrease homodimer or heterodimer formation and, therefore, are most likely to be useful as drugs for treating pathologies characterized by aberrant levels of apoptosis.